Immobilized enzymatic reactor

ABSTRACT

An immobilized enzymatic reactor can include a wall defining a chamber having an inlet and an outlet; a solid stationary phase covalently linked to an enzyme and disposed within the chamber; and a pressure modulator in fluid communication with the chamber and adapted to support continuous flow of a liquid sample comprising a polymer analyte through the inlet, over the solid stationary phase, and out of the outlet under a pressure between about 2,500 and 35,000 psi. In one example, the solid stationary phase includes inorganic/organic hybrid particles in an ultra performance liquid chromatography system, the enzyme is a protease, and the polymer analyte is a polypeptide. The immobilized enzymatic reactor can prepare an analyte for applications such as for hydrogen deuterium exchange mass spectrometry.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. provisionalpatent application No. 61/443,380 filed Feb. 16, 2011, and U.S.provisional patent application No. 61/474,155, filed Apr. 11, 2011,which are owned by the assignee of the instant applications and thedisclosures of which are incorporated by reference in their entirety.

FIELD OF THE TECHNOLOGY

The technology relates generally to immobilized enzymatic reactors(IMERs). The technology relates more particularly to high-pressure,continuous flow systems using a solid stationary phase covalently linkedto an enzyme in the analysis of liquid samples comprising a polymeranalyte.

BACKGROUND OF THE TECHNOLOGY

An immobilized enzyme is an enzyme that is attached to an insolublematerial, which allows enzymes to be held in place throughout areaction, separated from the products after the reaction, and used againif desired. There are three general methods for immobilizing an enzymein an IMER. The first general method includes adsorption on glass,alginate beads or matrix, in which the enzyme is attached to the outsideof an inert material. As adsorption is not a chemical reaction, theactive site of the immobilized enzyme may be blocked by the matrix orbead, reducing the activity of the enzyme. The second general methodincludes entrapment, in which the enzyme is trapped in insoluble beadsor microspheres, such as calcium alginate beads. However, the insolublesubstance can hinder the arrival of reactants and the exit of products.The third general method is cross-linkage, in which the enzyme iscovalently bonded to a matrix through a chemical reaction.

Covalently-linked IMERs and IMER media are available in various forms.For example, POROSZYME media, available from APPLIED BIOSYSTEMS(California, USA), employ polystyrenedivinylbenzene particles.STYROSZYME™ media, available from OraChrom, Inc. (Massachusetts, USA),employs fully pervious poly(styrene-divinylbenzene) matrices. Agarosebead media is also available from Thermo Fisher Scientific Inc(Massachusetts, USA). Presently, most commercial IMERs use these mediaunder low pressure conditions.

BRIEF SUMMARY OF THE TECHNOLOGY

The technology, in various embodiments, relates to apparatuses, methods,and kits for high-pressure, online/continuous flow IMER systems using asolid stationary phase covalently linked to an enzyme in the analysis ofliquid samples comprising a polymer analyte. The solid stationary phaseand system are adapted for high-pressure, continuous-flow operation,which can increase the efficiency and ease of use for the apparatuses,methods, and kits. In one example, the chamber can be an ultraperformance liquid chromatography (UPLC) column, the solid stationaryphase can be inorganic/organic hybrid particles (e.g., ethylene-bridged(BEH) particles), the enzyme can be a protease, the polymer analyte canbe a protein, and the system can support continuous flow of a liquidsample comprising the protein under a pressure between about 2,500 and15,000 psi. Thus, the apparatuses can prepare an analyte forapplications in proteomics such as for hydrogen deuterium exchange massspectrometry (HDX MS). Other examples and applications will beunderstood by persons of ordinary skill in the art from the disclosureand claims.

In one aspect, the technology features an apparatus that includes a walldefining a chamber having an inlet and an outlet, and a solid stationaryphase covalently linked to an enzyme and disposed within the chamber.The apparatus also includes a pressure modulator in fluid communicationwith the chamber and adapted to support continuous flow of a liquidsample comprising a polymer analyte through the inlet, over the solidstationary phase, and out of the outlet under a pressure between about2,500 and 35,000 psi.

In another aspect, the technology features a method that includescontinuously flowing a liquid sample comprising a polymer analyte over asolid stationary phase covalently linked to an enzyme, under a pressurebetween about 2,500 and 35,000 psi, thereby enzymatically cleaving thepolymer analyte.

In still another aspect, the technology features a method that includesselecting a solid stationary phase that can be linked to an enzyme andoperate under a pressure between about 2,500 and 35,000 psi. The methodalso includes disposing the solid stationary phase, covalently linked toan enzyme, within a chamber having an inlet and an outlet. The chamberis in fluid communication with a pressure modulator adapted to supportcontinuous flow of a liquid sample comprising a polymer analyte throughthe inlet, over the solid stationary phase, and out of the outlet underthe pressure.

In various embodiments, the solid stationary phase can includeinorganic/organic hybrid particles. The solid stationary phase caninclude ethylene-bridged (BEH) particles.

In some embodiments, the solid stationary phase can include a monolith,porous material, superficially porous material, porous particles, orsuperficially porous particles. The solid stationary phase can includepores having a mean pore volume within the range of 0.1-2.5 cm³/g. Thesolid stationary phase can include pores having a mean pore diameterwithin the range of 100-1000 Angstroms. The solid stationary phase canbe nonporous. The solid stationary phase can include ceramic, silica,inorganic silica, surface hybrid, or metal oxide. The solid stationaryphase can include particles having a mean size within the range of0.1-10 microns.

In certain embodiments, the covalent linker can include anorganofunctionalized silane linker. The covalent linker can betriethoxysilylbutyraldehyde.

In various embodiments, the pressure used in the apparatus or in one ormore of the methods is within the range of 8,000-15,000 psi.

In some embodiments, the apparatus can include one or more of thefollowing: a trapping column configured to collect the analyte flowingout of the outlet; a chromatography column configured to separate thecollected analyte; and a mass spectrometer configured to analyze theseparated analyte.

In certain embodiments, one or more of the methods can include one ormore of the following: collecting analyte flowing out of the outlet;separating the collected analyte by liquid chromatography; and analyzingthe separated analyte by mass spectrometry.

In various embodiments, one or more of the methods includes cycling thepressure between (i) the pressure between about 2,500 and 35,000 psi and(ii) a second pressure below about 2,500, while continuously flowing theliquid sample.

In some embodiments, the solid stationary phase is also linked to acapture group. The capture group can be a high affinity/specific captureagent (e.g., an antibody or antibody conjugate) or a lowaffinity/nonspecific capture agent. The capture group can be covalentlylinked to the solid stationary phase.

Online, high-pressure methods according to the technology haveadvantages including simpler operation and sample preparation thanconventional methods, as well as increased throughput. Such online, highpressure methods also have advantages including improved proteindigestions efficiency and the ability to digest polymers that areresistant to digestion at lower pressures. A polymer analyte can be abiopolymer such as a proteins, polypeptide, carbohydrate,deoxyribonucleic acid, or ribonucleic acid. A polymer analyte can be anon-natural polymer (hetero or homopolymer) having a linkage that can becleaved by an enzyme.

Other aspects and advantages of the technology will become apparent fromthe following drawings and description, all of which illustrateprinciples of the technology, by way of example only.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The advantages of the technology described above, together with furtheradvantages, may be better understood by referring to the followingdescription taken in conjunction with the accompanying drawings. Thedrawings are not necessarily to scale, emphasis instead generally beingplaced upon illustrating the principles of the technology.

FIG. 1 shows an example solid stationary phase preparation.

FIGS. 2A-C each shows a schematic of an example IMER system.

FIG. 3 shows a comparison of a 900 psi protein digestion (FIG. 3A) and a9,500 psi protein digestion (FIG. 3B).

FIG. 4 shows a comparison of the number of peptide fragments producedfrom phosphorylase b under 900 psi and 9500 psi.

FIG. 5 shows a comparison of % back-exchange in a fully deuteratedpolypeptide in a POROS® column and a BEH column.

DETAILED DESCRIPTION OF THE TECHNOLOGY

The technology includes apparatuses, methods, and kits forhigh-pressure, continuous flow (e.g., online) IMER systems using a solidstationary phase covalently linked to an enzyme in the analysis ofliquid samples comprising a polymer analyte. Such online methods haveadvantages including simpler operation and sample preparation thanconventional methods. Such high pressure methods also have advantagesincluding improved protein digestions efficiency and the ability todigest polymers that are resistant to digestion at lower pressures. Apolymer analyte can be a biopolymer such as a proteins, polypeptide,carbohydrate, deoxyribonucleic acid, or ribonucleic acid. A polymeranalyte can be a non-natural polymer (hetero or homopolymer) having alinkage that can be cleaved by an enzyme.

IMERs have numerous applications, for example in proteomics andbiotechnology. One common application for IMER systems is proteolysis,which is an important sample preparation step in proteincharacterization (e.g., prior to analysis, for example, by LC-MS).Protein digestion is generally performed offline by contacting aprotease and a sample protein, to cleave the sample protein into smallerfractionated peptides. Offline protein digestion is a standard protocol,but can have undesirable complications such as modification andreduction of the polypeptides. Other disadvantages of offline digestioninclude higher operator time and higher enzyme use (e.g., higher cost),when compared to methods according to the technology. Although proteindigestion is provided as an illustrative example, the technology can beapplied to essentially any analyte preparation or analysis that employsan enzymatic process.

Protein digestion can also be performed in an IMER. Commerciallyavailable IMER media include 20 μm POROSZYME media(polystyrenedivinylbenzene particles), available from APPLIED BIOSYSTEMSCalifornia, USA, STYROSZYME™ media (fully perviouspoly(styrene-divinylbenzene) matrices, available from OraChrom, Inc.,Massachusetts, USA), and agarose bead media. However, these media canonly be used at relatively low pressure (e.g., below 2,000 psi) becausehigher pressures will destroy the media and render the IMER inoperative.Therefore, such media are fundamentally limited in their applicationsand operating conditions.

In contrast to the prior art, the technology employs a solid stationaryphase adapted for online operation under high pressure. High pressurecan include a pressure between about 2,500 and 35,000 psi, for example,a pressure within the range of 8,000-15,000 psi. One example of apressure-resistant solid support is BEH particles. Modification on thesolid support can be achieved with a linker, e.g., an organofunctionalsilane chemical linker such as triethoxysilylbutyraldehyde, which canthen be used to immobilize a protease. This solid stationary phase canbe packed in a column to produce an IMER, which can then be used todigest proteins in an on-line LC system. On-line digestion can beperformed under high pressure, for example, in a Waters Corporation(Massachusetts, USA) UPLC-MS system for hydrogen deuterium exchange massspectrometry (HDX MS).

Importantly, solid stationary phase or support is not destroyed orrendered non-functional under high pressure operation. The technologyalso supports on-line operation (e.g., operation under pressure whilecontinuously flowing a sample over the solid support, as opposed tooff-line methods such as separate sample preparation or staticpressurized columns with sample cycling). On-line operation can increaseefficiency by allowing the fractionated peptides to be collected in atrapping column and then separated by a chromatograph in a rapid andcontinuous manner. Likewise, high pressure operation can also increaseefficiency, for example, by increasing the efficiency of proteolyticdigestions, improving analytical measurements, and increasing the rateof the operation of the device.

DEFINITIONS

As used above, the term “aliphatic group” includes organic compoundscharacterized by straight or branched chains, typically having between 1and 22 carbon atoms.

Aliphatic groups include alkyl groups, alkenyl groups and alkynylgroups. In complex structures, the chains can be branched orcross-linked. Alkyl groups include saturated hydrocarbons having one ormore carbon atoms, including straight-chain alkyl groups andbranched-chain alkyl groups. Such hydrocarbon moieties may besubstituted on one or more carbons with, for example, a halogen, ahydroxyl, a thiol, an amino, an alkoxy, an alkylcarboxy, an alkylthio,or a nitro group. Unless the number of carbons is otherwise specified,“lower aliphatic” as used herein means an aliphatic group, as definedabove (e.g., lower alkyl, lower alkenyl, lower alkynyl), but having fromone to six carbon atoms. Representative of such lower aliphatic groups,e.g., lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl,2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert-butyl,3-thiopentyl and the like. As used herein, the term “nitro” means —NO₂;the term “halogen” designates —F, —Cl, —Br or —I; the term “thiol” meansSH; and the term “hydroxyl” means —OH. Thus, the term “alkylamino” asused herein means an alkyl group, as defined above, having an aminogroup attached thereto. Suitable alkylamino groups include groups having1 to about 12 carbon atoms, or from 1 to about 6 carbon atoms. The term“alkylthio” refers to an alkyl group, as defined above, having asulfhydryl group attached thereto. Suitable alkylthio groups includegroups having 1 to about 12 carbon atoms, or from 1 to about 6 carbonatoms. The term “alkylcarboxyl” as used herein means an alkyl group, asdefined above, having a carboxyl group attached thereto. The term“alkoxy” as used herein means an alkyl group, as defined above, havingan oxygen atom attached thereto. Representative alkoxy groups includegroups having 1 to about 12 carbon atoms, or 1 to about 6 carbon atoms,e.g., methoxy, ethoxy, propoxy, tert-butoxy and the like. The terms“alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogousto alkyls, but which contain at least one double or triple bondrespectively. Suitable alkenyl and alkynyl groups include groups having2 to about 12 carbon atoms, or from 1 to about 6 carbon atoms.

The term “alicyclic group” includes closed ring structures of three ormore carbon atoms. Alicyclic groups include cycloparaffins or naphtheneswhich are saturated cyclic hydrocarbons, cycloolefins, which areunsaturated with two or more double bonds, and cycloacetylenes whichhave a triple bond. They do not include aromatic groups. Examples ofcycloparaffins include cyclopropane, cyclohexane and cyclopentane.Examples of cycloolefins include cyclopentadiene and cyclooctatetraene.Alicyclic groups also include fused ring structures and substitutedalicyclic groups such as alkyl substituted alicyclic groups. In theinstance of the alicyclics such substituents can further comprise alower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a loweralkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, orthe like.

The term “heterocyclic group” includes closed ring structures in whichone or more of the atoms in the ring is an element other than carbon,for example, nitrogen, sulfur, or oxygen. Heterocyclic groups can besaturated or unsaturated and heterocyclic groups such as pyrrole andfuran can have aromatic character. They include fused ring structuressuch as quinoline and isoquinoline. Other examples of heterocyclicgroups include pyridine and purine. Heterocyclic groups can also besubstituted at one or more constituent atoms with, for example, ahalogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a loweralkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, —CN, or the like. Suitable heteroaromatic andheteroalicyclic groups generally will have 1 to 3 separate or fusedrings with 3 to about 8 members per ring and one or more N, 0 or Satoms, e.g. coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl,furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl,piperidinyl, morpholino and pyrrolidinyl.

The term “aromatic group” includes unsaturated cyclic hydrocarbonscontaining one or more rings. Aromatic groups include 5- and 6-memberedsingle-ring groups which may include from zero to four heteroatoms, forexample, benzene, pyrrole, furan, thiophene, imidazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine and the like. The aromatic ring may be substituted at one ormore ring positions with, for example, a halogen, a lower alkyl, a loweralkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a loweralkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, or the like.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups and cycloalkylsubstituted alkyl groups. In certain embodiments, a straight chain orbranched chain alkyl has 30 or fewer carbon atoms in its backbone, e.g.,C₁-C₃₀ for straight chain or C₃-C₃₀ for branched chain. In certainembodiments, a straight chain or branched chain alkyl has 20 or fewercarbon atoms in its backbone, e.g., C₁-C₂₀ for straight chain or C₃-C₂₀for branched chain, and in some embodiments 18 or fewer. Likewise,particular cycloalkyls have from 4-10 carbon atoms in their ringstructure and in some embodiments have 4-7 carbon atoms in the ringstructure. The term “lower alkyl” refers to alkyl groups having from 1to 6 carbons in the chain and to cycloalkyls having from 3 to 6 carbonsin the ring structure.

Moreover, the term “alkyl” (including “lower alkyl”) as used throughoutthe specification and claims includes both “unsubstituted alkyls” and“substituted alkyls,” the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It willbe understood by those skilled in the art that the moieties substitutedon the hydrocarbon chain can themselves be substituted, if appropriate.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “aralkyl” moiety is an alkyl substituted with anaryl, e.g., having 1 to 3 separate or fused rings and from 6 to about 18carbon ring atoms, e.g., phenylmethyl (benzyl).

The term “aryl” includes 5- and 6-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example,unsubstituted or substituted benzene, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine and pyrimidine and the like. Aryl groups also includepolycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl andthe like. The aromatic ring can be substituted at one or more ringpositions with such substituents, e.g., as described above for alkylgroups. Suitable aryl groups include unsubstituted and substitutedphenyl groups. The term “aryloxy” as used herein means an aryl group, asdefined above, having an oxygen atom attached thereto.

The term “aralkoxy” as used herein means an aralkyl group, as definedabove, having an oxygen atom attached thereto. Suitable aralkoxy groupshave 1 to 3 separate or fused rings and from 6 to about 18 carbon ringatoms, e.g., O-benzyl.

The term “amino,” as used herein, refers to an unsubstituted orsubstituted moiety of the formula —NR_(a)R_(b), in which R_(a) and R_(b)are each independently hydrogen, alkyl, aryl, or heterocyclyl, or R_(a)and R_(b), taken together with the nitrogen atom to which they areattached, form a cyclic moiety having from 3 to 8 atoms in the ring.Thus, the term “amino” includes cyclic amino moieties such aspiperidinyl or pyrrolidinyl groups, unless otherwise stated. An“amino-substituted amino group” refers to an amino group in which atleast one of R_(a) and R_(b), is further substituted with an aminogroup.

The term “protecting group,” as used herein, refers to chemicalmodification of functional groups that are well known in the field oforganic synthesis. Exemplary protecting groups can vary, and aregenerally described in Protective Groups in Organic Synthesis [T. W.Green and P. G. M. Wuts, John Wiley & Sons, Inc, 1999].

“Hybrid,” including “organic-inorganic hybrid material,” includesinorganic-based structures wherein an organic functionality is integralto both the internal or “skeletal” inorganic structure as well as thehybrid material surface. The inorganic portion of the hybrid materialmay be, e.g., alumina, silica, titanium, cerium, or zirconium or oxidesthereof, or ceramic material. As noted above, exemplary hybrid materialsare shown in U.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035, 7,175,913and 7,919,177, the disclosures of which are hereby incorporated in theirentirety.

The term “BEH,” as used herein, refers to an organic-inorganic hybridmaterial which is an ethylene bridged hybrid material.

The term “adsorbed group,” as used herein, represents a monomer,oligimer or polymer, crosslinked or non-crosslinked that isnon-covalently attached to the core material. In certain embodiments,wherein Z represents an adsorbed group, the group can be adsorbed ontothe core material, X, the surface of the core material, X, or thesurface of the stationary phase material. Examples include, but are notlimited to alcohols, amines, thiols, polyamines, dedrimers, or polymers.

The term “functionalizing group” or “functionalizable group” includesorganic functional groups which impart a certain chromatographicfunctionality to a stationary phase.

The term “terminal group,” as used herein, represents a group whichcannot undergo further reactions. In certain embodiments, a terminalgroup may be a hydrophilic terminal group. Hydrophilic terminal groupsinclude, but are not limited to, protected or deprotected forms of analcohol, diol, glycidyl ether, epoxy, triol, polyol, pentaerythritol,pentaerythritol ethoxylate, 1,3-dioxane-5,5-dimethanol,tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethanepolyglycol ether, ethylene glycol, propylene glycol, poly(ethyleneglycol), poly(propylene glycol), a mono-valent, divalent, or polyvalentcarbohydrate group, a multi-antennary carbohydrate, a dendrimercontaining peripheral hydrophilic groups, a dendrigraph containingperipheral hydrophilic groups, or a zwitterion group.

The term “surface attachment group,” as used herein, represents a groupwhich may be reacted to covalently bond, non-covalently bond, adsorb, orotherwise attach to the core material, the surface of the core material,or the surface of the stationary phase material. In certain embodiments,the surface attachment group is attached to the surface of the corematerial by a siloxane bond. Surface attachment groups can providecovalent linkage between a solid stationary phase and an enzyme.

Solid Stationary Phases

IMER apparatuses, methods, and kits according to the technology canemploy various solid stationary phases, linkers, and enzymes to achievehigh-pressure, continuous flow operation and analysis of liquid samplescomprising a polymer analyte. One example is BEH (e.g., WatersCorporation, Massachusetts, USA, BEH 130 Angstrom, BEH 200 Angstrom, orBEH 300 Angstrom). Pressure can mean actual chamber pressure (e.g., asopposed to pressure per mm of solid stationary phase bed).

In general, the solid stationary phase can include essentially anymaterial selected and/or adaptable for high pressure IMER operation andcovalent linkage to an enzyme. High pressure can include, for example, apressure above about 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500,3,750, 4,000, 4,250, 4,500, 4,750, 5,000, 5,250, 5,500, 5,750, or 6,000psi or a pressure up to about 5,000, 6,000, 7,000, 8,000, 9,000 10,000,11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000,20,000, 22,500, 25,000, 27,500, 30,000, 32,500, or 35,000 psi or anyindividual value or range therebetween. Higher pressures are alsocontemplated by the technology. For example, pressures up to about40,000, 45,000, 50,000, 75,000, 100,000, 125,000, 150,000 or greater. Ingeneral, pressure should be selected in view of the solid support,instrument system, and desired flow rate. Covalent linkage to an enzymemay be achieved through a functional group on the surface of the solidstationary phase or through a linker molecule.

In various embodiments, the solid stationary phase can includeinorganic/organic hybrid particles. One example inorganic/organic hybridis ethylene-bridged (BEH) particles (Waters Corporation, Massachusetts,USA). The solid stationary phase can include a monolith, particles,porous particles, and/or superficially porous particles. Particles canbe spherical or non-spherical. The solid stationary phase can includesilica, inorganic silica, and/or metal oxide. In some embodiments, thechamber is equipped with one or more frits to contain the stationaryphase material. In embodiments in which the stationary phase material ismonolithic, the housing may be used without the inclusion of one or morefrits.

The solid stationary phase can include, for example, particles having amean size within the range of 1-10 microns, though a smaller or largersize could be selected if appropriate for a desired application. Invarious examples, the mean particle size is 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8, 8.6,9, 9.5, or 10 microns. In general, particle size can be selected in viewof the desired pressure and/or flow rate. For example, larger particlesize can be used to achieve consistent pressure from a column head to anend during high pressurized digestion. The solid stationary phase caninclude pores having a mean pore volume within the range of 0.1-2.5cm³/g. In various examples, the mean pore volume is 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 cm³/g. In some embodiments, porousparticles may be advantageous because they provide a relatively largesurface area (per unit mass or column volume) for protein coverage atthe same time as the ability to withstand high pressure.

The solid stationary phase can include pores having a mean pore diameterwithin the range of 100-1000 Angstroms. For example, the mean porediameter can be about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,or any value or range therebetween.

In certain embodiments, said stationary phase material comprisesparticles or a monolith having a core composition and a surfacecomposition represented by Formula 1:

W—[X]-Q  Formula 1

where X is core composition having a surface comprising a silica corematerial, a metal oxide core material, an organic-inorganic hybrid corematerial or a group of block polymers thereof; W is hydrogen orhydroxyl; and Q is absent or is a functional group that minimizeselectrostatic interactions, Van der Waals interactions, Hydrogen-bondinginteractions or other interactions with an analyte.

Furthermore, in certain embodiments, W and Q occupy free valences of thecore composition, X, or the surface of the core composition. In someembodiments, W and Q are selected to form a surface composition. Inother embodiments, X may be selected to form a block polymer or group ofblock polymers.

In certain embodiments, X is silica, titanium oxide, aluminum oxide oran organic-inorganic hybrid core comprising an aliphatic bridged silane.In specific embodiments, X is an organic-inorganic hybrid corecomprising a aliphatic bridged silane. In certain other specificembodiments, the aliphatic group of the aliphatic bridged silane isethylene.

In certain embodiments, the core material, X, may be cerium oxide,zirconium oxides, or a ceramic material. In certain other embodiments,the core material, X, may have a chromatographically enhancing poregeometry (CEPG). CEPG includes the geometry, which has been found toenhance the chromatographic separation ability of the material, e.g., asdistinguished from other chromatographic media in the art. For example,a geometry can be formed, selected or constructed, and variousproperties and/or factors can be used to determine whether thechromatographic separations ability of the material has been “enhanced,”e.g., as compared to a geometry known or conventionally used in the art.Examples of these factors include high separation efficiency, longercolumn life and high mass transfer properties (as evidenced by, e.g.,reduced band spreading and good peak shape). These properties can bemeasured or observed using art-recognized techniques. For example, thechromatographically-enhancing pore geometry of the present porousinorganic/organic hybrid particles is distinguished from the prior artparticles by the absence of “ink bottle” or “shell shaped” pore geometryor morphology, both of which are undesirable because they, e.g., reducemass transfer rates, leading to lower efficiencies.Chromatographically-enhancing pore geometry is found in hybrid materialscontaining only a small population of micropores. A small population ofmicropores is achieved in hybrid materials when all pores of a diameterof about <34 angstroms contribute less than about 110 m²/g to thespecific surface area of the material. Hybrid materials with such a lowmicropore surface area (MSA) give chromatographic enhancements includinghigh separation efficiency and good mass transfer properties (asevidenced by, e.g., reduced band spreading and good peak shape).Micropore surface area (MSA) is defined as the surface area in poreswith diameters less than or equal to 34 angstroms, determined bymultipoint nitrogen sorption analysis from the adsorption leg of theisotherm using the Barrett-Joyner-Halenda (BJH)method. As used herein,the acronyms “MSA” and “MPA” are used interchangeably to denote “micropore surface area.”

In certain embodiments the core material, X, may be surface modifiedwith a surface modifier having the formula Z′_(a)(R′)_(b)Si—R,″ whereZ′═Cl, Br, I, C₁-C₅ alkoxy, dialkylamino or trifluoromethanesulfonate; aand b are each an integer from 0 to 3 provided that a+b=3; R′ is a C₁-C₆straight, cyclic or branched alkyl group, and R″ is a functionalizinggroup. In another embodiment, the core material, X, may be surfacemodified by coating with a polymer.

In certain embodiments, the surface modifier is selected from the groupconsisting of octyltrichlorosilane, octadecyltrichlorosilane,octyldimethylchlorosilane and octadecyldimethylchlorosilane. In someembodiments, the surface modifier is selected from the group consistingof octyltrichlorosilane and octadecyltrichlorosilane. In otherembodiments, the surface modifier is selected from the group consistingof an isocyanate or 1,1′-carbonyldiimidazole (particularly when thehybrid group contains a (CH₂)₃OH group).

In another embodiment, the material has been surface modified by acombination of organic group and silanol group modification. In stillanother embodiment, the material has been surface modified by acombination of organic group modification and coating with a polymer. Ina further embodiment, the organic group comprises a chiral moiety. Inyet another embodiment, the material has been surface modified by acombination of silanol group modification and coating with a polymer.

In other embodiments, the material has been surface modified viaformation of an organic covalent bond between an organic group on thematerial and the modifying reagent. In still other embodiments, thematerial has been surface modified by a combination of organic groupmodification, silanol group modification and coating with a polymer. Inanother embodiment, the material has been surface modified by silanolgroup modification. In certain embodiments, the surface modified layermay be porous or nonporous.

In other embodiments of the stationary phase material, Q is ahydrophilic group, a hydrophobic group or absent. In some embodiments ofthe stationary phase material, wherein Q is a hydrophilic group, Q is analiphatic group. In other embodiments, said aliphatic group is analiphatic diol. In still other embodiments, Q is represented by Formula2:

wherein n¹ an integer from 0-30; n² an integer from 0-30; eachoccurrence of R¹, R², R³ and R⁴ independently represents hydrogen,fluoro, lower alkyl, a protected or deprotected alcohol, a zwiterion, ora group Z; Z represents:a) a surface attachment group produced by formation of covalent ornon-covalent bond between the surface of the stationary phase materialwith a moiety of Formula 3:

(B¹)_(x)(R⁵)_(y)(R⁶)_(z)Si—  Formula 3

-   -   wherein        -   x is an integer from 1-3,        -   y is an integer from 0-2,        -   z is an integer from 0-2,        -   and x+y+z=3    -   each occurrence of R⁵ and R⁶ independently represents methyl,        ethyl, n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl,        substituted or unsubstituted aryl, cyclic alkyl, branched alkyl,        lower alkyl, a protected or deprotected alcohol, or a zwiterion        group;    -   B¹ represents —OR⁷, —NR^(7′)R^(7″), —OSO₂CF₃, or —Cl; where each        of R⁷′ R^(7′) and R^(7″) represents hydrogen, methyl, ethyl,        n-butyl, iso-butyl, tert-butyl, iso-propyl, thexyl, phenyl,        branched alkyl or lower alkyl;        b) a direct attachment to a surface hybrid group of X through a        direct carbon-carbon bond formation or through a heteroatom,        ester, ether, thioether, amine, amide, imide, urea, carbonate,        carbamate, heterocycle, triazole, or urethane linkage; or        c) an adsorbed group that is not covalently attached to the        surface of the stationary phase material;        d) a surface attachment group produced by formation of a        covalent bond between the surface of the stationary phase        material, when W is hydrogen, by reaction with a vinyl or        alkynyl group;        Y represents a direct bond; a heteroatom linkage; an ester        linkage; an ether linkage; a thioether linkage; an amine        linkage; an amide linkage; an imide linkage; a urea linkage; a        thiourea linkage; a carbonate linkage; a carbamate linkage; a        heterocycle linkage; a triazole linkage; a urethane linkage; a        diol linkage; a polyol linkage; an oligomer of styrene, ethylene        glycol, or propylene glycol; a polymer of styrene, ethylene        glycol, or propylene glycol; a carbohydrate group, a        multi-antennary carbohydrates, a dendrimer or dendrigraphs, or a        zwitterion group; and        A represents    -   i.) a hydrophilic terminal group;    -   ii.) hydrogen, fluoro, fluoroalkyl, lower alkyl, or group Z; or    -   iii.) a functionalizable group.

In certain embodiments, wherein Q is an aliphatic diol of Formula 2, n¹an integer from 2-18, or from 2-6. In other embodiments, wherein Q is analiphatic diol of Formula 2, n² an integer from 0-18 or from 0-6. Instill other embodiments, wherein Q is an aliphatic diol of Formula 2, n¹an integer from 2-18 and n² an integer from 0-18, n¹ an integer from 2-6and wherein n² an integer from 0-18, n¹ an integer from 2-18 and n² aninteger from 0-6, or n¹ an integer from 2-6 and n² an integer from 0-6

In yet other embodiments of the stationary phase material, wherein Q isan aliphatic diol of Formula 2, A represents i) a hydrophilic terminalgroup and said hydrophilic terminal group is a protected or deprotectedforms of an alcohol, diol, glycidyl ether, epoxy, triol, polyol,pentaerythritol, pentaerythritol ethoxylate, 1,3-dioxane-5,5-dimethanol,tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethanepolyglycol ether, ethylene glycol, propylene glycol, poly(ethyleneglycol), poly(propylene glycol), a mono-valent, divalent, or polyvalentcarbohydrate group, a multi-antennary carbohydrate, a dendrimercontaining peripheral hydrophilic groups, a dendrigraph containingperipheral hydrophilic groups, or a zwitterion group.

In still other embodiments of the stationary phase material, wherein Qis an aliphatic diol of Formula 2, A represents ii.) hydrogen, fluoro,methyl, ethyl, n-butyl, t-butyl, i-propyl, lower alkyl, or group Z.

In still yet other embodiments of the stationary phase material, whereinQ is an aliphatic diol of Formula 2, A represents iii.) afunctionalizable group, and said functionalizable group is a protectedor deprotected form of an amine, alcohol, silane, alkene, thiol, azide,or alkyne. In some embodiments, said functionalizable group can giverise to a new surface group in a subsequent reaction step wherein saidreaction step is coupling, metathesis, radical addition,hydrosilylation, condensation, click, or polymerization.

In still other embodiments, the group Q can be a surface modifier.Non-limiting examples of surface modifiers that can be employed forthese materials include:

A.) Silanes that result in a hydrophollic surface modification

Hydrophillic Surface

Op- tion B¹ R⁵ R⁶ x/y/z n¹ 1 chloro, — — 3/0/0 3 methoxy, or ethoxy 2chloro, methyl, ethyl, — 2/1/0 3 methoxy, or n-propyl, i-pro- ethoxypyl, or t-butyl 3 chloro, methyl, ethyl, — 1/2/0 3 methoxy, or n-propyl,i-pro- ethoxy pyl, or t-butyl 4 chloro, methyl, ethyl, methyl, ethyl,1/1/1 3 methoxy, or n-propyl, i-pro- n-propyl, i-pro- ethoxy pyl, ort-butyl pyl, or t-butylWhere A is selected from the following:

or

B) silanes that result in a hydrophobic or a mixedhydrophollic/hydrophobic surface modification

Hydrophobic Surface

Op- tion B¹ R⁵ R⁶ x/y/z n¹ 1 chloro, — — 3/0/0 1-18 methoxy, or ethoxy 2chloro, methyl, ethyl, — 2/1/0 3-18 methoxy, or n-propyl, i-pro- ethoxypyl, or t-butyl 3 chloro, methyl, ethyl, — 1/2/0 1-3- methoxy, orn-propyl, i-pro- 18 ethoxy pyl, or t-butyl 4 chloro, methyl, ethyl,methyl, ethyl, 1/1/1 3-18 methoxy, or n-propyl, i-pro- n-propyl, i-pro-ethoxy pyl, or t-butyl pyl, or t-butylWhere A is selected from the following; H, phenyl, NHC(O)NHR⁸, NHC(O)R⁸,OC(O)NHR⁸, OC(O)OR⁸, or triazole-R⁸, where R⁸ is octadecyl, dodecyl,decyl, octyl, hexyl, n-butyl, t-butyl, n-propyl, i-propyl, phenyl,benzyl, phenethyl, phenylethyl, phenylpropyl, diphenylethyl, biphenylyl.

In certain embodiments, Z represents an attachment to a surfaceorganofunctional hybrid group through a direct carbon-carbon bondformation or through a heteroatom, ester, ether, thioether, amine,amide, imide, urea, carbonate, carbamate, heterocycle, triazole, orurethane linkage.

In other embodiments, Z represents an adsorbed, surface group that isnot covalently attached to the surface of the material. This surfacegroup can be a cross-linked polymer, or other adsorbed surface group.Examples include, but are not limited to alcohols, amines, thiols,polyamines, dedrimers, or polymers.

Enzymes

IMER apparatuses, methods, and kits according to the technology canemploy various enzymes to achieve high-pressure, continuous flowoperation and analysis of liquid samples comprising a polymer analyte.One example is pepsin, which is employed in HDX MS for samplepreparation.

In general, the enzyme can include essentially any protein catalyst thatcan function for sample preparation or analysis. One example enzyme is aprotease. A protease can be a specific cleavage protease (e.g., trypsin)or a non-specific cleavage protease (e.g., pepsin). The technology canbe used with numerous different types of enzyme such as trypsin,chymotrypsin, lysC, amidases, PNGase, PNGase F, V8, as well as mixturesof these and/or other enzymes, in various proteomics applications. Aprotease can be selected from any one of the following broad groups:Serine proteases, Threonine proteases, Cysteine proteases, Aspartateproteases, Metalloproteases, and Glutamic acid proteases. Alternatively,protease can be selected from any one of the following broad groups:Acid proteases, Neutral proteases, or Basic proteases (or alkalineproteases). The enzyme can include a combination of more than oneprotease (e.g., having cumulative and/or complementary functions). Theenzyme (or enzymes) can be selected to achieve a desired function (e.g.,cleaving one or more specific bonds) and/or operate under desiredconditions (e.g., matching an enzyme's operating pH to the pH of asample).

In addition to the enzyme, the solid stationary phase can be linked to acapture group. The capture group can be a high affinity/specific captureagent (e.g., an antibody or antibody conjugate) or a lowaffinity/nonspecific capture agent. The capture group can be covalentlylinked to the solid stationary phase. The capture group can provide asecond mode of action (e.g., increase the resonance time of the polymeranalyte, thereby increasing the efficiency of the IMER).

Covalent Linkers

IMER apparatuses, methods, and kits according to the technology canemploy various linkers to immobilize an enzyme of interest on a solidstationary phases. One example is Triethoxysilylbutyraldehyde(bALD-TEOS, available from Gelest, Inc.)

In general, the covalent linker can include essentially any chemicalmoiety that can covalently connect the solid stationary phase and theenzyme (e.g., an amide bond formed by a carboxylic acid and an amine).Other examples included amines and NHS-aldehydes such as succinimidylp-formylbenzoate (SFB) or succinimidyl p-formylphenoxyacetate (SFPA), aswell as amines and glutaraldehydes. In certain embodiments, the covalentlinker can include an organofunctionalized silane linker. Theorganofunctionalized silane linker can be triethoxysilylbutyraldehyde.In general, enzymes can be covalently joined to a linker through Schiffbase formation and reductive amination. In general, silane linkers canbe covalently joined to a solid support having hydroxyl groups throughcontacting the linker and solid support, and quenching the reactionmixture with ethanolamine. Other enzymes can be immobilized on a solidsupport such as BEH using an appropriate immobilization protocol (e.g.,selected for the chemical functionality of the solid support and bindingsite on the protein). Depending on the enzymatic activity at a targetedpH, a different pH may be required during immobilization protocol. Forexample, a BEH trypsin column may need to be prepared at a neutral pHrather than an acidic pH.

FIG. 1 shows an example solid stationary phase preparation reaction.First, an enzyme (pepsin) is reacted with a linker(Triethoxysilylbutyraldehyde, bALD-TEOS) through Schiff base formationand reductive amination by NaCNBH₃. Second, enzyme-linker is reactedwith alcohol groups on the surface of the solid support (acid treatedBEH) and quenched with ethanolamine.

bALD-TEOS is an aldehyde compound that forms Schiff Base with a primaryor secondary amine (NH—). Every protein contains the N-terminus with afree amine, thus the N-terminus of a pepsin and any available side-chainamine groups are subjected to form a Schiff Base conjugate withbALD-TEOS.

The ALD coupling solution (Sterogene Bioseparation, Inc.) was added intothe conjugate mixture as a mild reductant (NaCNBH₃) to initiate thereductive amination. Once the Schiff base is reduced through an agitatedincubation for 2 hours, this type of linkage becomes stable.

Acid treated BEH (5 micron diameter, 300 Angstrom pore size) was addedinto the solution containing the reduced conjugate of bALD-TEOS andpepsin. The other end of bALD-TEOS contains the triethoxysily groupsavailable to react with hydroxyl group of silanol on the surface of BEHparticles. The reaction was completed after overnight rotation, and thenquenched by adding 1M ethanolamine to block the unreacted aldehydegroups.

The batches of pepsin BEH were washed with Na₃Citrate, NaCl, and finallywashed and stored in 0.08% TFA in water. Pepsin is irreversiblydeactivated under basic conditions. Thus the pH of the pepsin solutionwas kept acidic under pH 5 throughout the batch synthesis.

The solid stationary phase linked to the enzyme can then be placed in achamber (e.g., a column such as the 2.1×30 mm UPLC housing with 0.1%formic acid in water) for use in an IMER system. In various embodiments,the chamber can be an UPLC housing. However, in general, the chamberneed only be adapted for online, high pressure operation and may assumea variety of geometric configurations (e.g., short and squat for lowerresonance times or long and thin for longer resonance times).

High Pressure Systems

IMERs can be used under high pressure, for example, in high pressure LCsystems. One example is the nanoACQUITY UltraPerformance LC® (UPLC®)system available from Waters Corporation (Massachusetts, USA) combinedwith Waters Corporation SYNAPT™ Q-TOF™ mass spectrometry and acquired inelevated-energy mass spectrometry (MSE) mode and processed using aWaters Corporation ProteinLynx Global SERVER™ (PLGS, which is a fullyintegrated Mass-Informatics™ platform for quantitative and qualitativeproteomics research).

FIGS. 2A-C show schematics of exemplary on-line IMER systems. A pepsincolumn was prepared by the method shown in FIG. 1, using pepsin,bALD-TEOS, and BEH 5 micron, 300 Angstrom particles. The pepsin-BEHparticles were packed in UPLC column hardware (2.1×30 mm), which wasthen installed on a nanoACQUITY UltraPerformance LC®. The IMER systemwas set up and operated on-line for HDX-MS, which increased the speedand convenience of the analysis.

The IMER system components included two 6-port valves (i.e., each portlabeled 1-6 in the schematics shown in FIGS. 2A-C), which are shown inthe positions corresponding to the stage of experiments. 100 μL/min of0.05% formic acid pH 2.5 was flowed in auxiliary solvent manager throughpepsin column. The gradient at 40 μL/min was flowed in a binary solventmanager. In each case, the protein sample was loaded into pepsin columnand digested for approximately 30 seconds. FIGS. 2A and 2B show thesystem operating with the valve in trapping mode. FIG. 2A shows theexperimental setup for a low pressure (900 psi) and FIG. 2B shows theexperimental setup for a high pressure (9,500 psi). In the experimentshown in FIG. 2B, a 2.1×150 mm column was placed after a trappingcolumn, to function as a flow restrictor (e.g., a pressure modulator).

Although the example flow restrictor (e.g., pressure modulator) is showna 2.1×150 mm column was placed after a trapping column, flow resistorscan be essentially any structure or device that creates and/or maintainspressure in the system. For example, a flow resistor can be a capillarytubing, packed column, or microfluidic device.

A Waters Corporation (Massachusetts, USA) trapping column 201 (2.1×5 mmBEH C18, 1.7 micron diameter) was used to remove salts from the digestedpeptides in a desalting step. After the digestion and consequenttrapping (3 minutes), the valves were switched from trapping mode toeluting mode, and the peptides were separated in an analytical columnfor MS analysis.

FIG. 2C shows the IMER system with the valve in eluting mode. Trappedpeptides were released from trap column and separated in analyticalcolumn using a solvent gradient. During eluting mode, high pressure wasnot applied to the pepsin column. The solvent flow went through thepepsin column directed into waste. The binary solvent manager (BSM)drove the flow to the trapping column to elute the trapped peptides.Finally the peptides were separated in analytical column.

In another set of experiments, a trypsin column was used in place of thepepsin column in the inline IMER system shown in FIGS. 2A-C. With thetrypsin column, the auxiliary solvent manager (ASM) delivered 20 mMammonium bicarbonate at pH 7.9 with flow rate at 10 μL/min.

The apparatus and methods described in connection with FIGS. 2A-C wereused to digest multiple model proteins including phosphorylase b, bovineserum albumin, cytochrome C, and a monoclonal antibody. In each case,the online, high pressure IMER exhibited high digestion efficiency andexperimental reproducibility, as well as sequence coverage of up to 95%without deterioration of the solid stationary phase.

HDX MS is one example analytical technique for which the IMER system canbe used. In another example, tryptic IMER can be used as an onlinetryptic digestion device (e.g., as an alternative to widely used offlinetryptic digestion methods), which has various proteomics applications.In yet another example, the technology can be used for 2D LC onlinedigestion for protein identification and/or characterization. Inaddition to sample preparation methods, the technology can be used withvarious analytical methods including refractive index detectors, UVdetectors, light-scattering detectors and mass spectrometers.

Experimental Results

FIG. 3A shows a chromatogram of phosphorylase b after the 900 psi pepsindigestion described in connection with FIG. 2A. At 900 psi,phosphorylase b was digested to produce only 76% sequence coverage with118 common peptides. In contrast, FIG. 3B shows a chromatogram ofphosphorylase b after the 9,500 psi pepsin digestion described inconnection with FIG. 2B. At 9,500 psi, phosphorylase b was digested toproduce 82% sequence coverage with 139 common peptides, without anyintact protein remaining. Therefore, the higher pressure digestionresulted in a greater number of peptides and a higher sequence coverage.

FIG. 4. shows a plot of the number of peptides for each peptide lengthof 4-23, for the 900 psi digestions (light bar on the right hand side,corresponding to FIGS. 2A and 3A) and for the 9,500 psi digestion (darkbar on the left hand side, corresponding to FIGS. 2B and 3B). Again,this figure illustrates that a high pressure lead to a greater number ofshorter peptides (e.g., higher sequence coverage) in the digestion ofphosphorylase b.

In general, pressurized digestion is also advantageous because it canincrease digestion efficiency for proteins, including proteins that aredifficult to digest at low pressure. Furthermore, the improved sequencecoverage and digestion efficiency which results from the high-pressure,online apparatus and method of the technology is advantageous inproteomics applications such as HDX MS because it can provide improvedresolution of localized conformational changes in higher order proteinstructure.

During the HDX MS, a phenomena called “back-exchange” can occur: oncethe hydrogen from protein backbone is exchanged with deuterium, thelabeled deuterium can quickly go back to hydrogen when the temperature,pH, and pepsin column media are not properly controlled. Back-exchangecauses a loss of information on localized conformational changes inhigher order protein structures. Thus, it is important to achieve lowerlevel of back-exchange through fast digestion and/or rapid desalting(e.g., a less than 10 minute separation) in HDX MS.

FIG. 5. shows % back-exchange at various temperatures in an HDX MSanalysis of fully deuterated Bradykinin. The figure compares the %back-exchange in an embodiment of the present technology (an online,high pressure pepsin IMER setup described in FIG. 2B) and the %back-exchange from a low pressure POROS® pepsin column (a setup similarto FIG. 2A, commercially available from Applied Biosystems, California,USA). Both experiments in the comparison were performed with the sameflow rate, temperature, and reagents. The only variable was the solidstationary phase and the pressure—the Poros apparatus was operated atlow pressure and that apparatus according to an embodiment of thetechnology was operated online, at high pressure. At all temperaturesbelow 37° C., the embodiment of the present technology exhibited a lowerback-exchange rate than the POROS® column. Without intending to be boundby any particular theory, it is believed that the superior operation ofthe technology is due, at least in part, to the increased proteindigestion efficiency at high pressure.

The foregoing illustrations and examples demonstrate the advantages ofthe technology in the context of HDX MS—increased protein digestionspeed and efficiency, combined with reduced experimental artifacts andsignal loss (e.g., back-exchange). These and other advantages in variousproteomics and biotechnology application will be apparent to those ofordinary skill in the art.

While the technology has been particularly shown and described withreference to specific embodiments, it should be understood by thoseskilled in the art that various changes in form and detail may be madewithout departing from the spirit and scope of the technology as definedby the appended claims.

What is claimed is:
 1. An apparatus comprising: a wall defining achamber having an inlet and an outlet; a solid stationary phasecovalently linked to an enzyme and disposed within the chamber; and apressure modulator in fluid communication with the chamber and adaptedto support continuous flow of a liquid sample comprising a polymeranalyte through the inlet, over the solid stationary phase, and out ofthe outlet under a pressure between about 2,500 and 35,000 psi.
 2. Theapparatus of claim 1, wherein the solid stationary phase comprisesinorganic/organic hybrid particles.
 3. The apparatus of claim 1, whereinthe solid stationary phase comprises ethylene-bridged (BEH) particles.4. The apparatus of claim 1, wherein the solid stationary phasecomprises a monolith, porous material, superficially porous material,porous particles, superficially porous particles, or nonporous materialor particles.
 5. The apparatus of claim 1, wherein the solid stationaryphase comprises pores having a mean pore volume within the range of0.1-2.5 cm³/g.
 6. The apparatus of claim 1, wherein the solid stationaryphase comprises pores having a mean pore diameter within the range of100-1000 Angstroms.
 7. The apparatus of claim 1, wherein the solidstationary phase comprises ceramic, silica, inorganic silica, surfacehybrid, or metal oxide.
 8. The apparatus of claim 1, wherein the solidstationary phase comprises particles having a mean size within the rangeof 0.1-10 microns.
 9. The apparatus of claim 1, wherein the covalentlinker comprises an organofunctionalized silane linker.
 10. Theapparatus of claim 1, wherein the covalent linker comprisestriethoxysilylbutyraldehyde.
 11. The apparatus of claim 1, wherein thepressure is within the range of 8,000-15,000 psi.
 12. The apparatus ofclaim 1, further comprising: a trapping column configured to collect theanalyte flowing out of the outlet; a chromatography column configured toseparate the collected analyte; and a mass spectrometer configured toanalyze the separated analyte.
 13. The apparatus of claim 1, furthercomprising a capture group linked to the solid stationary phase.
 14. Amethod comprising: continuously flowing a liquid sample comprising apolymer analyte over a solid stationary phase covalently linked to anenzyme, under a pressure between about 2,500 and 35,000 psi, therebyenzymatically cleaving the polymer analyte.
 15. The method of claim 14,wherein the solid stationary phase comprises ethylene-bridged (BEH)particles.
 16. The method of claim 14, wherein the covalent linkercomprises triethoxysilylbutyraldehyde.
 17. The method of claim 14,wherein the pressure is within the range of 8,000-15,000 psi.
 18. Themethod of claim 14, further comprising: collecting analyte flowing outof the outlet; separating the collected analyte by liquidchromatography; and analyzing the separated analyte by massspectrometry.
 19. The method of claim 14, wherein the solid stationaryphase is linked to a capture group.
 20. A method comprising: selecting asolid stationary phase that can be linked to an enzyme and operate undera pressure between about 2,500 and 35,000 psi; disposing the solidstationary phase, covalently linked to an enzyme, within a chamberhaving an inlet and an outlet, the chamber in fluid communication with apressure modulator adapted to support continuous flow of a liquid samplecomprising a polymer analyte through the inlet, over the solidstationary phase, and out of the outlet under the pressure.
 21. Themethod of claim 20, wherein the solid stationary phase comprisesethylene-bridged (BEH) particles.
 22. The method of claim 20, whereinthe covalent linker comprises triethoxysilylbutyraldehyde.
 23. Themethod of claim 20, wherein the pressure is within the range of8,000-15,000 psi.
 24. The method of claim 20, further comprising cyclingthe pressure between (i) the pressure between about 2,500 and 35,000 psiand (ii) a second pressure below about 2,500, while continuously flowingthe liquid sample.
 25. The method of claim 20, wherein the solidstationary phase is linked to a capture group.